Expression Analysis Of Salinity-related Genes And Development Of SNP In Portunus Trituberculatus

The swimming crab, P. trituberculatus, is a commercially importantfishery species in China and widely distributed on sandy and muddybottoms in the coastal waters of Japan, Korea, China and Taiwan. It is aeuryhaline crab species, surviving in wide-range salinity conditions, andwater salinity condition influenced its artificial propagations significantly.Aquaporins (AQP) which belong to a major intrinsic proteins (MIP)family plays important roles in salinity stress response. In this study,aquaporins cDNA of P. trituberculatus was cloned by rapid amplification ofcDNA ends (RACE) and named PtAQP. The full-length of PtAQP cDNA is1712bp, including an open reading frame (ORF) of771bp which encodesa256amino acid polypeptide. Bioinformatics software analysis reavealedthat PtAQP gene contains6transmembrane domains,2NPA structuralunits and conservative amino acid sequence which is matched to MIPfamily. Homologous analysis showed that PtAQP of P. trituberculatus hasthe highest homology to AQP1of Callinectes sapidus. The quantitativereal-time RT-PCR showed that PtAQP gene could be detected in all testedtissues of P. trituberculatus, with the highest expression level in stomach.After challenged by high and low salinity, the expression of PtAQP gene ingill and hepatopancreas of P. trituberculatus first falling then rise. Ourresults confirmed the osmotic regulation function of PtAQP gene andrevealed the regulatory mechanism of PtAQP gene under salinity stress.Chloride channels are found on the plasma membrane of virtually alleukaryotic cells and play important roles in the maintenance of water andelectrolyte balance, in the regulation of cell volume, and in the control ofmembrane potential. Chloride intracellular channel (CLIC) gene is a kind of Chloride channels gene, and it plays important roles in salinity stressresponse. In this study, chloride intracellular channel cDNA of P.trituberculatus was cloned by rapid amplification of cDNA ends (RACE)and named PtCLIC. The full-length of PtCLIC cDNA is2000bp,including an open reading frame (ORF) of762bp which encodes a253amino acid polypeptide. Bioinformatics software analysis reavealed thatPtCLIC gene does not have transmembrane domains and belongs toinstability protein. Homologous analysis showed that PtCLIC of P.trituberculatus has the highest homology of83%to CLIC of Daphniapulex. Phylogenetic analysis showed that P. trituberculatus firstly cluster toScylla paramamosian. The quantitative real-time RT-PCR showed thatPtCLIC gene could be detected in all tested tissues of P. trituberculatus,with the highest expression level in hepatopancreas. After challenged bylow salinity, the expression of PtCLIC gene in gill and hepatopancreas of P.trituberculatus first up-regulated then down-regulated and the expression ofPtCLIC gene in low salinity tolerant and susceptive family showedsignificantly difference. Our results confirmed the osmotic regulationfunction of PtCLIC gene and can assist in breeding P. trituberculatus lowsalinity tolerant strain.Calreticulin (CRT) is a highly conserved and multifunctionalendoplasmic reticulum (ER) chaperone protein and plays important roles insalinity stress response. In order to research the function of calreticulinunder salinity stress, the full-length cDNA sequence of calreticulin from P.trituberculatus (PtCRT) was firstly cloned and characterized. The completecDNA sequence of PtCRT is1676bp with1218bp open reading frame(ORF), encoding a polypeptide of405amino acids. Multiple sequencealignments showed that the deduced acid amino sequences of PtCRTshared the highest homology to CRT of F. chinensis (89%). Fluorescentquantitative real-time PCR analysis indicated that PtCRT expressed in alldetected tissues and hepatopancreas showed the highest expression level. Inaddition, salinity challenge significantly converted the expression level ofPtCRT in gill and hepatopancreas. In the whole cDNA sequence, four single nucleotide polymorphisms (SNPs) were detected. All of them sitedin the coding region and none of them was associated with the tolerance/susceptibility of salinity challenge. All results indicated that PtCRT playsan important role in mediating the osmotic pressure of P. trituberculatus.